PROJECT SUMMARY Only 0.01% of cancer cells enter circulation, survive & produce metastasis, however, metastatic disease accounts for 90% of cancer-related deaths. In pancreatic ductal adenocarcinoma (PDA), the majority of patients present with extra-pancreatic invasion and metastatic disease for which the there is a dismal five-year survival rate and no specific therapeutic strategies directed at the treatment of metastatic disease. Notably, much of the research into the mechanisms of metastasis has been focused on identifying early drivers within primary tumors, however, the identification of central drivers of metastatic outgrowth within established lesions largely remain unexplored. Recently, we have demonstrated that SIRT6 inactivation dramatically accelerates PDA development, resulting in highly aggressive metastatic disease in the Kras-p53 GEM model. Employing this highly aggressive PDA metastasis model, in addition to an established breast cancer metastasis model, this study aims to identify potential vulnerabilities of metastatic lesions that could be exploited to treat patients with advanced metastatic disease. Performing unbiased RNA-Seq on matched primary and metastatic tissues followed by a newly developed 96-well soft agar screen, we identified factors that are uniquely required for anchorage-independent growth of established metastatic cells. Utilizing this functional screen and validation, we have identified Gstt1 (glutathione S-transferase theta 1) as a top candidate driver of metastatic outgrowth in multiple mouse models of metastasis. Preliminary data demonstrates that Gstt1 is differentially expressed in metastatic cell lines compared to matched primary-derived cell lines and inhibition of Gstt1 significantly reduced metastatic potential of metastases-derived cells, without affecting primary tumor growth, suggesting an important role for Gstt1 in the outgrowth of established metastatic lesions. Additionally, within metastatic lesions, Gstt1 shows a heterogenous expression pattern, where Gstt1high cells represent an aggressive, non-proliferative sub- population. Additionally, we have demonstrated that Gstt1 is required for the formation of tumor spheres in breast and PDA metastatic-derived cell lines in vitro, suggesting a role for Gstt1 as a driver of self-renewal in metastatic cells. In this proposal we seek to identify characteristics unique to Gstt1high PDA-derived metastatic lesions. The proposed studies will provide important preclinical demonstration of whether Gstt1 and its downstream targets are required for sustained growth of metastatic tumors, thus identifying a possible therapeutic window for this subset of metastatic PDA.